Srebp-1c/Fgf21/Pgc-1α Axis Regulated by Leptin Signaling in Adipocytes—Possible Mechanism of
3. Results
3.1. Role of SREBP-1c in the Effects of CR on Gene Expression in WAT
We have reported previously that CR increases the expression ofSrebp-1c,Srebp-1a, andPgc-1a mRNAs in the WAT of Wd B6;129S6 mice but not in KO mice [16]. In the WAT of mice on a C57Bl/6 background, CR also increased the expression ofSrebp-1c,Pgc-1a, andFgf21mRNAs in Wd mice but not in KO mice (Figure1A or Figure1C,D). Similar findings about the CR-associated upregulation of Fgf21mRNA were observed in B6;129S6 mice (Figure S2). However, in contrast to mice on a B6;129S6 background, CR did not upregulate the expression ofSrebp-1amRNA in either Wd or KO mice on a C57Bl/6 background (Figure1B). Overall, it is likely that the CR-associated upregulation of these factors is less exaggerated in C57bl/6 mice compared with B6;129S6 mice.
Figure 1.The effects ofSrebp-1cKO on the expression of key regulators of CR-associated metabolic remodeling in the WAT of mice on a C57Bl/6 background. The mRNA expression levels ofSrebp-1c(A), Srebp-1a(B),Fgf21(C), andPgc-1a(D) in WAT were measured using RT-PCR and were normalized to Tbpexpression (n=4). Values are means±SDs. *p<0.05, **p<0.01 vs. AL, according to Student’s t-test, or two-way ANOVA and Tukey’s test.
3.2. Effects of SREBP-1c on the Expression of Genes and Proteins Involved in FA Biosynthesis and Mitochondrial Biogenesis
To confirm that SREBP-1c is the significant regulator of the expression of genes and proteins involved in FA biosynthesis and mitochondrial biogenesis in vitro, we generated 3T3-L1 preadipocytes that overexpressed SREBP-1c (SREBP-1c OE) using retroviral vectors. The SREBP-1c OE 3T3-L1
preadipocytes were differentiated to adipocytes (Figure2A), and the expression levels of mRNAs and proteins of interest were analyzed.
Figure 2.The effects of SREBP-1c overexpression on the expression of genes and proteins involved in fatty acid (FA) biosynthesis and mitochondrial biogenesis in mature 3T3-L1 adipocytes. Control and SREBP-1c OE preadipocytes were differentiated into mature adipocytes in four separate dishes from each phenotype. RNA was extracted and lysates were prepared from each dish. RNA was extracted and lysates were prepared from adipocytes. The mRNA expression levels ofSrebp-1c(A),Srebp-1a(B), PeriA(C),Adipoq(D),Fasn(E),Fgf21(F), andPgc-1a(G) were determined using RT-PCR and normalized toRps18expression (n=4). (H) Representative immunoblot images, showing the expression levels of proteins involved in FA biosynthesis and mitochondrial biogenesis. Quantitative analysis was performed using a chemiluminescence method. The protein expression of ACC (I), ME-1 (J), FGF21 (K), PGC-1α(L), TFAM (M), and SIRT3 (N) are shown as the relative intensities of the indicated protein divided by that of LMNB1 as an internal control (n=4). Values are means±SDs. *p<0.05, **p<0.01,
***p<0.001 vs. controls, according to Student’st-test.
In SREBP-1c OE adipocytes, the expression ofSrebp-1amRNA was similar to that of control cells (Figure2B), whereas that ofperilipin A(PeriA) andadiponectin(Adipoq), which are markers of adipocyte differentiation, was upregulated (Figure2C,D). Fatty acid synthase (FASN) is a rate-limiting enzyme in FA biosynthesis. The expression ofFasnmRNA was high in SREBP-1c OE adipocytes (Figure2E).
Moreover, the expression of bothFgf21andPgc-1amRNAs was high in OE adipocytes (Figure2F,G).
In addition, the protein expression of acetyl-CoA carboxylase (ACC) and malic enzyme 1 (ME1), which are FA biosynthetic enzymes, and FGF21 and PGC-1αwere high in OE adipocytes (Figure2H–L).
With regard to mitochondrial proteins, expression of SIRT3 was also high in SREBP-1c OE adipocytes, but that of TFAM was not (Figure2H or Figure2M,N).
To further characterize the regulation of FA biosynthetic genes by FGF21 and PGC-1α, we generated Wd and KO adipocytes by differentiating MEFs derived from Wd andSrebp-1cKO mice. The expression ofSrebp-1cwas not detectable in KO adipocytes and that ofSrebp-1awas similar to that of control adipocytes (Figure3A,B). The expression levels ofPeriAandAdipoqmRNAs in KO adipocytes did not differ from those in Wd adipocytes, suggesting that SREBP-1c deficiency did not alter differentiation (Figure3C,D). However, the expression ofFasnwas lower in KO adipocytes than Wd adipocytes (Figure3E). The expression ofFgf21mRNA was slightly reduced, while that ofPgc-1amRNA was significantly lower in KO adipocytes (Figure3F,G). Taken together, our findings suggest that the expression of bothPgc-1aandFgf21is positively regulated by Srebp-1c, in addition to that ofFasn.
Moreover, SREBP-1c is the significant regulator of the expression of genes and proteins involved in FA biosynthesis and mitochondrial biogenesis in adipocytes.
Figure 3.The effects of SREBP-1c deficiency on the expression of adipocyte differentiation markers and genes involved in FA biosynthesis and mitochondrial biogenesis in mature adipocytes. MEFs were obtained from four individual embryos of either Wd orSrebp-1cKO mice, differentiated to mature adipocytes, and then RNA was extracted from each dish. The mRNA expression levels ofSrebp-1c(A), Srebp-1a(B),PeriA(C),Adipoq(D),Fasn(E),Fgf21(F), andPgc-1a(G) were determined using RT-PCR and normalized toRps18expression (n=4). Values are means±SDs. ***p<0.001 vs. Wd, according to Student’st-test.
3.3. Roles of FGF21 and PGC-1αin Mitochondrial Biogenesis
CR upregulated the expression of bothFgf21andPgc-1αmRNAs and proteins via SREBP-1c.
Therefore, we next determined the roles of FGF21 and PGC-1αin mitochondrial biogenesis.
In FGF21 OE adipocytes (Figure4A or Figure4C,D), the expression ofPgc-1αmRNA and PGC-1α protein was high (Figure4B,C or Figure4E,F). Treatment with PD173074, an FGF receptor (FGFR) inhibitor, reduced the phosphorylation of ERK without reducingFgf21mRNA and FGF21 protein expression (Figure4A or Figure4C–E). In addition, this treatment did not reduce the expression of Pgc-1αmRNA or PGC-1αprotein (Figure4B,C or Figure4F).
Figure 4.The effects of FGF21 overexpression and the inhibition of FGFR on the expression of genes and proteins involved in FGF21 signaling and PGC-1αin mature 3T3-L1 adipocytes. Control and FGF21 OE preadipocytes were differentiated into mature adipocytes in four separate dishes for each phenotype, and they were treated with or without 50 nM PD173074, an FGFR inhibitor, for 24 h.
(A,B) RNA was extracted and lysates were prepared from each dish. The mRNA expression levels of Fgf21(A) andPgc-1a(B) were determined using RT-PCR and normalized toRps18expression (n=4).
(C) Representative immunoblot images showing the expression of proteins involved in FGF21 signaling and mitochondrial biogenesis. Quantitative analysis was performed using a chemiluminescence method. The protein expression of FGF21 (D) and PGC-1α(F) is shown as the relative intensity of the indicated protein divided by that of LMNB1 as an internal control (n=4). Extracellular signal-regulated kinase (ERK) phosphorylation is expressed as the relative intensity of the phosphorylated form of ERK/total ERK (n=4) (E). Values are means±SDs *p<0.05, **p<0.01, ***p<0.001 vs. controls administered the same treatment.
In Fgf21 KO adipocytes differentiated from MEFs, the expression levels ofPeriA,Adipoq, andPgc-1α mRNAs were much lower than in control cells, suggesting that the significant reduction inPgc-1α mRNA expression is associated with impaired adipocyte differentiation (Figure5A–D).
Figure 5.The effects of FGF21 deficiency on the expression of adipocyte differentiation markers and genes involved in mitochondrial biogenesis in mature adipocytes. MEFs were obtained from four individual embryos of either Wd orFgf21KO mice, differentiated to mature adipocytes, and then RNA was extracted from each dish. The mRNA expression levels ofFgf21(A),PeriA(B),Adipoq(C), andPgc-1a(D) were analyzed using RT-PCR and normalized toRps18expression (n=4). Values are means±SDs. *p<0.05, **p<0.01 vs. Wd, according to Student’st-test, ***p<0.001.
Taken together, these findings indicate that FGF21 positively regulates PGC-1αexpression, but ERK signaling does not have a significant effect.
3.4. Effect of Leptin Signaling on the Expression of Genes and Proteins Involved in FA Biosynthesis and Mitochondrial Biogenesis
To determine the effect of leptin signaling on FA biosynthesis and mitochondrial biogenesis in adipocytes, we knocked down leptin receptor expression in 3T3-L1 preadipocytes using a retroviral vector and analyzed the cells after differentiating them to adipocytes (Figure6A). The activation of the leptin receptor phosphorylates STAT3, the major downstream target molecule of leptin signaling [38].
Leptin receptor knockdown (KD) reduced the phosphorylation of STAT3, confirming that leptin signaling had been inhibited (Figure6F,G). The expression ofSrebp-1c,Srebp-1a,Fgf21, andPgc-1α mRNAs was significantly higher in leptin receptor KD adipocytes than in control cells (Figure6B–E).
Moreover, the expression of FGF21, PGC-1α, TFAM, ACC, and ME-1 proteins was higher in leptin receptor KD adipocytes (Figure6F or Figure6H–L). This finding suggests that a reduction in leptin signaling induces the expression of bothSrebp-1candSrebp-1amRNAs and the expression of proteins involved in FA biosynthesis and mitochondrial biogenesis in adipocytes.
Figure 6.The effects of leptin signaling on the expression of genes and proteins involved in leptin signaling, FA biosynthesis, and mitochondrial biogenesis in mature 3T3-L1 adipocytes. LeptinR knockdown (KD) (shLeptinR) and control (shGFP) preadipocytes were differentiated into mature adipocytes in four separate dishes for each phenotype, and then RNA was extracted, and lysates were prepared from each dish. The mRNA expression levels ofLeptinR(A),Srebp-1a(C),Fgf21(D), andPgc-1a (E) were determined using RT-PCR and normalized toRps18expression (n=4). (B) Representative images of ethidium bromide-stained gels, showing fluorescence corresponding to the products of Srebp-1ccDNA amplification by RT-PCR. Semiquantitative analysis was performed and the data were normalized toRps18expression (n=4). (F) Representative immunoblot images showing the expression of proteins involved in leptin signaling, FA biosynthesis, and mitochondrial biogenesis. Quantitative analysis was performed using a chemiluminescence method. The protein expression of pSTAT (G), FGF21 (H), PGC-1α(I), TFAM (J), ACC (K), and ME-1 (L) is shown as the relative intensity of the indicated protein divided by that of LMNB1 as an internal control (n=4). Values are means±SDs. *p
<0.05, **p<0.01, ***p<0.001 vs. shGFP, according to Student’st-test.